Method for producing L-methionine by fermentation

ABSTRACT

L-Methionine is produced by culturing a microorganism which is deficient in repressor of L-methionine biosynthesis system and/or enhanced intracellular homoserine transsuccinylase activity is cultured in a medium so that L-methionine should be produced and accumulated in the medium, and collecting the L-methionine from the medium. The microorganism preferably further exhibits reduced intracellular S-adenosylmethionine synthetase activity, L-threonine auxotrophy, enhanced intracellular cystathionine γ-synthase activity and enhanced intracellular aspartokinase-homoserine dehydrogenase II activity. The present invention enables breeding of L-methionine-producing bacteria, and L-methionine production by fermentation.

TECHNICAL FIELD

The present invention relates to a method for producing L-methionine byfermentation. L-methionine is an important amino acid as a medicamentand the like.

BACKGROUND ART

Industrially produced methionine mainly consists of DL-methionine, whichis produced through chemical synthesis. When L-methionine is required,it is provided through production of N-acetyl-DL-methionine byacetylation of DL-methionine and subsequent enzymatic selectivedeacetylation of the N-acetylated L-methionine.

On the other hand, as for the production of L-methionine byfermentation, methods utilizing an L-methionine analogue-resistantmutant strain have been reported. However, their production amount issmall, and factors affecting the L-methionine production have not beenelucidated yet. Therefore, L-methionine is still one of the amino acidsthe most difficult to be produced by fermentation. For example, whilemethods utilizing Escherichia coli (E. coli) K-12 strain have beenreported in Japanese Patent Laid-open (Kokai) No. 56-35992 andliterature (Chattapadhyay, M. K. et al., Med. Sci. Res. 23, 775 (1995);Chattapadhyay, M. K. et al., Biotechnol. Lett. 17, 567-570 (1995)), anyof these methods cannot provide L-methionine production amountsufficient for industrial use.

In E. coli, the biosynthetic pathway of L-methionine is partly sharedwith the biosynthetic pathway of L-threonine, and L-homoserine serves asa common intermediate. The first step of the peculiar pathway fromL-homoserine to L-methionine is catalyzed by homoserine transsuccinylase(HTS). This enzyme has been known to suffer concerted inhibition by thefinal product, L-methionine, and a metabolite of L-methionine,S-adenosylmethionine (Lee, L.-W. et al., J. Biol. Chem., 241, 5479-5480(1966)).

The nucleotide sequence of the metA gene encoding homoserinetranssuccinylase of E. coli, has been reported by Duclos et al. (Duclos,B. et al., Nucleic Acids Res. 17, 2856 (1989)), and a method forobtaining a strain having a mutation for metA using resistance to ananalog of L-methionine, α-methyl-DL-methionine (MM) has also been known(Chattopadhyay, M. K. et al., J. Gen. Microbiol., 137, 685-691 (1991)).It has been reported for Salmonella typhimurium that, the metA geneproduct, homoserine transsuccinylase, was an inhibition-desensitizedtype as for the inhibition by L-methionine and S-adenosylmethioninesynthase (SAM) in an MM resistant strain (Lawrence, D. A. et al., J.Bacteriol., 109, 8-11 (1972)). However, the nucleotide sequence of themutant meta gene has not been reported. Furthermore, it has beenreported that a mutant having a sole mutation in metA did not secretL-methionine (Chattopadhyay, M. K. et al., J. Gen. Microbiol., 137,685-691 (1991)).

It has also been revealed that the expression of the genes includingmetA of the enzymes for the reaction by homoserine transsuccinylase andsubsequent reactions in the peculiar biosynthetic pathway ofL-methionine suffers inhibition by a repressor which is a metJ geneproduct (Green, R. C. Biosynthesis of Methionine in “Escherichia coliand Salmonella Cellular and Molecular Biology/Second Edition”, ed.Neidhardt, F. D., ASM Press, pp. 542-560 (1996)). It has also been knownthat the metJ gene is adjacent to the metBL operon in a reversedirection, which operon consists of the metB gene coding for the secondenzyme of the peculiar biosynthetic pathway for L-methionine,cystathionine γ-synthase, and metL coding for aspartokinase-homoserinedehydrogenase II (AK-HDII) (Duchange, N. et al., J. Biol. Chem., 258,14868-14871 (1983)).

It has been suggested that metK coding for S-adenosylmethionine, whichcatalyzes the metabolic reaction from L-methionine toS-adenosylmethionine, should be an essential enzyme (Green, R. C.Biosynthesis of Methionine in “Escherichia coli and Salmonella Cellularand Molecular Biology/Second Edition”, ed. Neidhardt, F. D., ASM Press,pp. 542-560 (1996)). Furthermore, it has also been known that a metKmutant strain can be obtained based on resistance to a methionineanalogue such as DL-norleucine and ethionine (Chattopadhyay, M. K. etal., J. Gen. Microbiol., 137, 685-691 (1991)), and can increase theexpression of the enzymes of the peculiar biosynthetic pathway ofL-methionine (Greene, R. C. et al., J. Bacteriol., 115, 57-67).

As mentioned above, there have been reported enzymes involved in theL-methionine biosynthesis and genes therefor to some extent. However,only few findings that directly lead to the production of L-methionineby fermentation have been obtained, and hence hardly applied to breedingof L-methionine-producing bacteria.

SUMMARY OF THE INVENTION

The present invention has been accomplished in view of theaforementioned current technical status, and an object of the presentinvention is to elucidate factors affecting the L-methionine production,thereby breeding L-methionine-producing bacteria, and enablingL-methionine production by fermentation.

In order to achieve the aforementioned object, the present inventorsearnestly conducted studies, and as a result, accomplished the presentinvention.

That is, the present invention provides:

(1) A microorganism which is deficient in repressor of L-methioninebiosynthesis system and has L-methionine productivity;

(2) a microorganism having enhanced intracellular homoserinetranssuccinylase activity and L-methionine productivity;

(3) a microorganism which is deficient in repressor of L-methioninebiosynthesis system, and has enhanced intracellular homoserinetranssuccinylase activity and L-methionine productivity;

(4) the microorganism according to any one of the above (1)-(3), whichfurther exhibits reduced intracellular S-adenosylmethionine synthetaseactivity;

(5) the microorganism according to any one of the above (2)-(4), whereinthe enhanced intracellular homoserine transsuccinylase activity isobtained by increasing copy number of a gene encoding homoserinetranssuccinylase, or enhancing an expression regulatory sequence for thegene;

(6) the microorganism according to the above (1) or (4), which hashomoserine transsuccinylase for which concerted inhibition byL-methionine and S-adenosylmethionine is desensitized;

(7) the microorganism according to any one of the above (1)-(6), whichexhibits L-threonine auxotrophy;

(8) the microorganism according to any one of the above (1)-(7), whichexhibits enhanced intracellular cystathionine γ-synthase activity andenhanced intracellular aspartokinase-homoserine dehydrogenase IIactivity;

(9) the microorganism according to any one of the above (1)-(8), whichbelongs to the genus Escherichia;

(10) a method for producing L-methionine which comprises culturing themicroorganism according to any one of the above (1)-(9) in a medium toproduce and accumulate L-methionine in the medium, and collecting theL-methionine from the medium; and

(11) A DNA which codes for homoserine transsuccinylase for whichconcerted inhibition by L-methionine and S-adenosylmethionine isdesensitized, wherein the homoserine transsuccinylase has the amino acidsequence of SEQ ID NO: 26 including a mutation corresponding toreplacement of arginine by cysteine at the 27th position, mutationcorresponding to replacement of isoleucine by serine at the 296thposition, mutation corresponding to replacement of proline by leucine atthe 298th position, mutation corresponding to replacement of arginine bycysteine at the 27th position and replacement of isoleucine by serine atthe 296th position, mutation corresponding to replacement of isoleucineby serine at the 296th position and replacement of proline by leucine atthe 298th position, mutation corresponding to replacement of proline byleucine at the 298th position and replacement of arginine by cysteine atthe 27th position, or mutation corresponding to replacement of arginineby cysteine at the 27th position, replacement of isoleucine by serine atthe 296th position and replacement of proline by leucine at the 298thposition.

In this specification, S-adenosylmethionine will occasionally beabbreviated as “SAM”, α-methyl-DL-methionine as “MM”, and DL-norleucineas “NL”. Further, S-adenosylmethionine synthetase will be occasionallybe abbreviated as “SAM synthetase”, and homoserine transsuccinylase as“HTS”. The metB gene product, cystathionine γ-synthase, of E. coli mayalso be called as “cystathionine synthase”, and the metL gene product,“aspartokinase homoserine dehydrogenase II”, may also be called asAK-HDII.

The term “L-methionine productivity” used for the present inventionmeans an ability to accumulate L-methionine in a medium when amicroorganism is cultured in the medium.

According to the present invention, there is provided a microorganismhaving L-methionine production ability. The microorganism can beutilized as an L-methionine-producing bacterium or a material forbreeding of L-methionine-producing bacteria.

The mutant metA gene of the present invention can be utilized for thebreeding of L-methionine-producing bacteria, because the concertedinhibition by L-methionine and SAM for the enzyme encoded by it iscanceled.

DETAILED DESCRIPTION OF THE INVENTION

Hereafter, the present invention will be explained in detail.

The microorganism of the present invention is a microorganism which isdeficient in repressor of the L-methionine biosynthesis system and hasL-methionine productivity, or a microorganism which has enhancedintracellular homoserine transsuccinylase activity and L-methionineproductivity. The microorganism of the present invention is preferably amicroorganism which is deficient in repressor of the L-methioninebiosynthesis system, and has enhanced intracellular homoserinetranssuccinylase activity. The microorganism of the present inventionfurther preferably exhibits reduced intracellular SAM synthetaseactivity.

The aforementioned microorganism of the present invention is notparticularly limited, so long as it has a pathway for producingL-methionine and SAM from L-homoserine via O-acylhomoserine which isproduced from L-homoserine by the acyl-transferring reaction, and itsexpression of the acyl transferase is controlled through suppression bya repressor. While such a microorganism may be an Escherichia bacterium,coryneform bacterium, and Bacillus bacterium, it is preferably anEscherichia bacterium, for example, E. coli.

If the microorganism of the present invention is a bacterium in whichHTS possessed by the microorganism suffers concerted inhibition by SAMand L-methionine like E. coli, its L-methionine productivity may beimproved by canceling the inhibition.

As the peculiar pathway for the methionine biosynthesis, there are onewhich uses cystathionine as an intermediate as in many microorganismssuch as E. coli, and one which does not use cystathionine as inBrevibacterium flavum (Ozaki, H. et al., J. Biochem., 91, 1163 (1982)).In the present invention, a microorganism that uses cystathionine ispreferred. In such a microorganism, L-methionine productivity can beincreased by enhancing the intracellular cystathionine synthaseactivity. In addition, even if it is a microorganism like Brevibacteriumflavum, L-methionine productivity may be enhanced by deficiency ofrepressor in the L-methionine biosynthesis system and/or enhancement ofHTS.

Furthermore, in the aforementioned microorganism, L-methionineproductivity can further be increased by enhancing at least one of theaspartokinase activity and the homoserine dehydrogenase activity, whichare involved in the shared pathway of the L-methionine biosynthesis andthe L-threonine biosynthesis.

When two or more of the aforementioned characteristics are imparted to amicroorganism, the order for imparting them is not particularly limited,and they can be given in an arbitrary order. Moreover, when multiplegenes are introduced into a microorganism, those genes may be carried bythe same vector, or may be separately carried by multiple differentvectors. When multiple vectors are used, it is preferred to use vectorshaving different drug markers and different replication origins.

Methods for imparting each of the aforementioned characteristics to amicroorganism will be explained below.

<1> Deficiency of Repressor in L-Methionine Biosynthesis System

A microorganism deficient in a repressor in the L-methioninebiosynthesis system can be obtained by subjecting microorganisms to amutagenic treatment, and selecting a strain no longer producing therepressor. The mutagenic treatment can be performed with means usuallyused for obtaining microbial mutants, for example, UV irradiation ortreatment with an agent used for mutagenesis such asN-methyl-N′-nitrosoguanidine (NTG) and nitrous acid.

The repressor can also be made deficient by destroying a gene coding forthe repressor on chromosomal DNA of the microorganism. The gene can bedestroyed by preparing a deleted type gene which has deletion of atleast a part of a coding region or expression regulatory sequence, andcausing homologous recombination of the deleted type gene and a gene onthe chromosome to substitute the deleted type gene for the gene on thechromosome (gene substitution).

Since the nucleotide sequence of the gene coding for the repressor inthe L-methionine biosynthesis system of E. coli (metJ) has been known(Duchange, N. et al., J. Biol. Chem., 258, 14868-14871 (1983)), therepressor can be isolated from chromosomal DNA, for example, by PCRusing primers produced based on the nucleotide sequence. A deleted typegene can be obtained by excising a certain region from the gene fragmentobtained as described above, and deleting at least a part of the codingregion or expression regulatory region.

The gene substitution can be performed, for example, as follows. Adeleted type gene is introduced into a vector having a temperaturesensitive replication origin to prepare a recombinant vector, and amicroorganism is transformed with the recombinant vector so that thedeleted type gene should be inserted into a gene on chromosomal DNA byhomologous recombination of the deleted type gene and the gene on thechromosomal DNA. Then, the transformant strain is cultured at atemperature at which the vector cannot replicate to drop out the vectorfrom cytoplasm. Furthermore, the gene is replaced when one copy of thegene on the chromosome is dropped out with the vector. The occurrence ofthe desired gene substitution can be confirmed by Southern hybridizationanalysis of the chromosomal DNA of a strain to be to be tested for thegene substitution.

As a vector for E. coli that has a temperature sensitivity replicationorigin, for example, the plasmid pMAN997 disclosed in Japanese PatentApplication No. 9-194603 and the like can be mentioned. As a vector forcoryneform bacteria that has a temperature sensitivity replicationorigin, for example, the plasmid pHSC4 disclosed in Japanese PatentLaid-open No. 5-7491 and the like can be mentioned. However, the vectoris not limited to these, and other vectors can also be used.

As mentioned above, it has known for E. coli that the metJ gene isadjacent to the metBL operon, which consists of metB gene and metL gene,in the reverse direction (Duchange, N. et al., J. Biol. Chem., 258,14868-14871 (1983)). Therefore, if a suitable promoter sequence isligated to a deleted type metJ gene and gene substitution is performedas described above, the destruction of the metJ gene and improvement ofexpression utilizing substitution of promoter in the metBL operon cansimultaneously be obtained by one homologous recombination. Improvedexpression of the metBL operon enhances the intracellular cystathioninesynthase activity and AK-HDII activity.

Specifically, the following three components, a fragment of about 1 kbcontaining the metB gene, which is obtained by, for example, PCR(polymerase chain reaction; White, T. J. et al.; Trends Genet., 5, 185(1989)) utilizing chromosomal DNA of E. coli W3110 at strain as atemplate, and oligonucleotides having the nucleotide sequences of SEQ IDNO: 5 and SEQ ID NO: 6 as primers; a fragment of about 1 kb containingthe downstream region of the metJ gene obtained by PCR utilizingoligonucleotides having the nucleotide sequences of SEQ ID NO: 7 and SEQID NO: 8 as primers; and a sequence having a promoter sequence of thethreonine operon, which is obtained by annealing of the oligonucleotidesrepresented as SEQ ID NO: 9 and SEQ ID NO: 10, can be inserted into asuitable vector, and ligating to it to obtain a recombinant vector whichcontains a DNA fragment having deletion of the structural gene of metJand substitution of threonine promoter for the promoter of the metBLoperon.

To introduce the recombinant DNA prepared as described above tobacterium, any known transformation methods can be employed. Forinstance, employable are a method of treating recipient cells withcalcium chloride so as to increase the permeability of DNA, which hasbeen reported for Escherichia coli K-12 [see Mandel, M. and Higa, A., J.Mol. Biol., 53, 159 (1970)]; and a method of preparing competent cellsfrom cells which are at the growth phase followed by introducing the DNAthereinto, which has been reported for Bacillus subtilis [see Duncan, C.H., Wilson, G. A. and Young, F. E., Gene, 1, 153 (1977)]. Alternatively,it is also possible to apply a method in which DNA recipient cells areallowed to be in a state of protoplasts or spheroplasts capable ofincorporating recombinant DNA with ease to introduce recombinant DNAinto the DNA recipient cells, as known for Bacillus subtilis,actinomycetes, and yeasts (Chang, S, and Choen, S, N., Molec. Gen.Genet., 168, 111 (1979); Bibb, M. J., Ward, J. M. and Hopwood, O. A.,Nature, 274, 398 (1978); Hinnen, A., Hicks, J. B. and Fink, G. R., Proc.Natl. Acad. Sci. USA, 75, 1929 (1978)). Transformation of coryneformbacteria may performed by the electric pulse method (refer to JapanesePatent Publication Laid-Open No. 2-207791).

The vector to be used for cloning the genes such as metA, metK and thrBCas described below includes, for example, pUC19, pUC18, pBR322, pHSG299,pHSG399, pHSG398, RSF1010 and the like. Besides, it is possible to usephage DNA vectors. When microorganisms other than E. coli are used, itis preferable to use a shuttle vector autonomously replicable in thosemicroorganisms and E. coli. As examples of plasmid autonomouslyreplicable in coryneform bacteria, for example, the followings can bementioned.

pAM330 (see Japanese Patent Laid-open No. 58-67699)

pHM1519 (see Japanese Patent Laid-open No. 58-77895)

pAJ655 (see Japanese Patent Laid-open No. 58-192900)

pAJ611 (see the same)

pAJ1844 (see the same)

pCG1 (see Japanese Patent Laid-open No. 57-134500)

pCG2 (see Japanese Patent Laid-open No. 58-35197)

pCG4 (see Japanese Patent Laid-open No. 57-183799)

pCG11 (see the same)

pHK4 (see Japanese Patent Laid-open No. 5-7491)

In order to prepare recombinant DNA by ligating the gene fragment and avector, the vector is digested by restriction enzyme(s) corresponding tothe termini of the gene fragment. Ligation is generally performed byusing a ligase such as T4 DNA ligase.

The methods to perform, for example, preparation of the genomic DNAlibrary, hybridization, PCR, preparation of plasmid DNA, digestion andligation of DNA, and design of oligonucleotide used for primers aredescribed by Sambrook, J., Fritsche, E. F., Maniatis, T. in MolecularCloning, Cold Spring Harbor Laboratory Press (1989).

<2> Enhancement of HTS Activity and Introduction of Mutant HTS

The HTS activity in a microbial cell can be attained by preparing arecombinant DNA through ligation of a gene fragment encoding HTS with avector which functions in the microorganism, preferably a multi-copytype vector, and transforming the microorganism through introduction ofthe plasmid into in the microbial cell. As a result of increase of thecopy number of the gene encoding HTS in the transformant strain, the HTSactivity is enhanced. In E. coli, HTS is encoded by the metA gene. Whenan Escherichia bacterium is used as the microorganism, the HTS gene tobe introduced is preferably a gene derived from an Escherichiabacterium. However, genes derived from other microorganisms havinghomoserine transacetylase, such as coryneform bacteria, can also beused.

Enhancement of HTS activity can also be achieved by introducing multiplecopies of the HTS gene into the chromosomal DNA of the above-describedhost strains. In order to introduce multiple copies of the HTS gene inthe chromosomal DNA of bacterium belonging to the genus Corynebacterium,the homologous recombination is carried out using a sequence whosemultiple copies exist in the chromosomal DNA as targets. As sequenceswhose multiple copies exist in the chromosomal DNA, repetitive DNA,inverted repeats exist at the end of a transposable element can be used.Also, as disclosed in Japanese Patent Laid-open No. 2-109985, it ispossible to incorporate the HTS gene into transposon, and allow it to betransferred to introduce multiple copies of the HTS gene into thechromosomal DNA. By either method, the number of copies of the HTS genewithin cells of the transformant strain increases, and as a result, HTSactivity is enhanced.

The enhancement of HTS activity can also be obtained by, besides beingbased on the aforementioned gene enhancement, enhancing an expressionregulatory sequence for the HTS gene. Specifically, it can be attainedby replacing an expression regulatory sequence of HTS gene on chromosomeDNA or plasmid, such as a promoter, with a stronger one (see JapanesePatent Laid-open No. 1-215280). For example, lac promoter, trc promoter,tac promoter, PR promoter and PL promoter of lambda phage and the likeare known as strong promoters. Substitution of these promoters enhancesexpression of the HTS gene, and hence the HTS activity is enhanced.

Since the nucleotide sequence of the HTS gene (metA) of E. coli has beenknown (Blattner, F. R. et al., Science, 277, 1453-1462 (1997)), it canbe isolated from chromosomal DNA by PCR using primers produced based onthe nucleotide sequence. As such primers, the oligonucleotides havingthe nucleotide sequences represented as SEQ ID NO: 21 and SEQ ID NO: 22are specifically mentioned.

It is expected that, by enhancing the HTS activity in a microbial cellas described above, L-methionine biosynthesis can be enhanced, and thusthe L-methionine production amount can be increased.

Further, because HTS suffers concerted inhibition by L-methionine andSAM, the L-methionine biosynthesis system can also be enhanced byobtaining a microorganism containing HTS for which concerted inhibitionhas been canceled. Such a microorganism containing HTS for whichconcerted inhibition has been canceled can be obtained by subjectingmicroorganisms to a mutagenic treatment, and selecting a straincontaining HTS for which concerted inhibition has been canceled. Themutagenic treatment can be performed with means usually used forobtaining microbial mutants, for example, UV irradiation or treatmentwith an agent used for mutagenesis such as N-methyl-N′-nitrosoguanidine(NTG) and nitrous acid. The expression “HTS for which concertedinhibition has been canceled” herein used means HTS exhibiting a ratioof its enzymatic activity in the presence of L-methionine, SAM orL-methionine and SAM to its enzymatic activity in the absence ofL-methionine and SAM (remaining ratio) higher than that of a wild-typeHTS. Specifically, an HTS exhibiting a remaining ratio in the presenceof 1 mM L-methionine of 40% or more, preferably 80% or more, a remainingratio in the presence of 1 mM SAM of 10% or more, preferably 50% ormore, or a remaining ratio in the presence of 0.1 mM each ofL-methionine and SAM of 15% or more, preferably 60% or more is an HTSfor which concerted inhibition by L-methionine and SAM has beencanceled.

A mutant strain having such a mutant HTS as mentioned above can beobtained by culturing a parent strain in the presence ofα-methyl-DL-methionine (MM), e.g., in a medium containing 1 g/l of MM,and selecting a strain growing on the medium. The selection with MM maybe repeated two or more times.

A mutant strain having a mutant HTS can also be obtained by cloning themutant HTS gene (mutant metA) from a HTS mutant obtained as describedabove, and transforming a microorganism with the mutant gene. Isolationof a mutant HTS gene and introduction of the gene into a microorganismcan be performed as the aforementioned wild-type HTS gene. As HTS havinga mutant metA gene, HTS having the amino acid sequence of SEQ ID NO: 26including a mutation corresponding to replacement of arginine bycysteine at the 27th position, mutation corresponding to replacement ofisoleucine by serine at the 296th position, or mutation corresponding toreplacement of proline by leucine at the 298th position can specificallybe mentioned. HTS including two or more of these mutations is also apreferred mutant HTS.

<3> Attenuation of SAM Synthetase Activity

Furthermore, the L-methionine productivity of microorganism can beincreased by attenuating intracellular SAM synthetase activity. TheL-methionine productivity of a microorganism can also be increased bymaking the microorganism SAM synthetase activity deficient, but in sucha case, the medium for culturing the microorganism must contain SAM.Therefore, it is preferable to attenuate SAM synthetase activity. Theexpression “attenuating SAM synthetase activity” herein used means thata specific activity of SAM synthetase per unit of protein in microbialcells is made lower than that of a strain having a wild-type SAMsynthetase. Specifically, the degree of attenuation, i.e., the reducedactivity may be 80% to 50%, preferably 50% to 30%, more preferably 30%to 10% of the SAM synthetase activity of a wild-type strain. In E. coli,it has been suggested that, if the specific activity of SAM synthetasefalls below 10%, cell division would be inhibited (Newman, E. B. et al.,J. Bacteriol., 180, 3614-3619 (1998)).

The microorganism whose SAM synthetase activity is reduced may be oneproducing SAM synthetase exhibiting a reduced specific activity perenzymatic protein (reduced type SAM synthetase), or one in whichexpression efficiency of the enzyme is reduced because of reducedtranscription efficiency or reduced translation efficiency of SAMsynthetase gene.

A mutant strain whose SAM synthetase activity is reduced may be obtainedby culturing a parent strain in the presence of DL-norleucine (NL),e.g., in a medium containing 0.1 g/l of NL, and selecting a grownstrain. The selection with NL may be repeated two or more times. It isalso possible to use ethionine or γ-glutamylmethyl ester instead ofDL-norleucine.

A mutant strain having an reduced type SAM synthetase can also beobtained by cloning a gene for the reduced type SAM synthetase from aSAM synthetase-reduced strain obtained as described above, andsubstituting the mutant gene for a wild-type SAM synthetase gene on achromosome of microorganism. The gene substitution of SAM synthetasegene can be performed in the same manner as the aforementioned metJgene. Since the nucleotide sequence of the SAM synthetase gene (metK) ofE. coli has been known (Blattner, F. R. et al., Science, 277, 1453-1462(1997)), it can be isolated from chromosomal DNA by PCR using primersproduced based on the nucleotide sequence. As such primers, theoligonucleotides having the nucleotide sequences represented as SEQ IDNO: 11 and SEQ ID NO: 12 are specifically mentioned. The occurrence ofthe desired mutation in the obtained metK gene can be confirmed bydetermining the nucleotide sequence of the gene, and comparing it with aknown nucleotide sequence of wild-type metK gene.

As specific examples of the gene coding for an reduced type SAMsynthetase, those coding for SAM synthetases having the amino acidsequence of SEQ ID NO: 18 including a mutation corresponding toreplacement of isoleucine by leucine at the 303rd position, mutationcorresponding to replacement of valine by glutamic acid at the 185thposition, or mutation corresponding to replacement of arginine at the378th position and subsequent amino acid residues by a sequence ofalanine-methionine-leucine-proline-valine (SEQ ID NO: 29) can bementioned.

<4> L-Threonine Auxotrophy

L-methionine productivity can be improved by imparting L-threonineauxotrophy to a microorganism. Specific examples of a microorganismexhibiting L-threonine auxotrophy include those having deficiency of anyone of enzymes involved in the peculiar pathway of L-threoninebiosynthesis from L-homoserine to L-threonine. In E. coli, the genes ofthe enzymes involved in the biosynthesis of L-threonine exist as thethreonine operon (thrABC), and L-threonine auxotrophic strain, which haslost the ability to synthesize L-homoserine and subsequent products, canbe obtained by deleting the thrBC segment. The thrA gene codes for oneof the isozymes of aspartokinase, which is an enzyme of the sharedpathway of the L-methionine and L-threonine biosyntheses, and hence itis preferably not to be deleted.

In order to delete thrBC, the thrBC segment in the threonine operon onchromosomal DNA can be destroyed. thrBC can be destroyed by replacingthe thrBC segment on a microbial chromosome with thrBC a part of whichis deleted. The gene substitution of thrBC may be performed in the samemanner as in the gene substitution of the aforementioned metJ gene. AthrBC segment containing deletion can be obtained by amplifying afragment of about 1 kb containing the upstream region of the thrB geneby PCR using E. coli chromosomal DNA as a template and primers havingthe nucleotide sequences of SEQ ID NOS: 1 and 2, similarly amplifying afragment of about 1 kb containing the downstream region of the thrC geneby PCR using primers having the nucleotide sequences of SEQ ID NOS: 3and 4, and ligating these amplified products.

<5> Production of L-Methionine

L-methionine can be produced by culturing a microorganism havingL-methionine productivity prepared as described above in a medium sothat L-methionine should be produced and accumulated in the medium, andcollecting L-methionine from the medium.

The medium to be used may be selected from well-known mediaconventionally used depending on the kind of microorganism to be used.That is, it may be a usual medium that contains a carbon source,nitrogen source, inorganic ions, and other organic ingredients asrequired. Any special medium is not needed for the practice of thepresent invention.

As the carbon source, it is possible to use sugars such as glucose,lactose, galactose, fructose or starch hydrolysate; alcohols such asglycerol or sorbitol; or organic acids such as fumaric acid, citric acidor succinic acid.

As the nitrogen source, it is possible to use inorganic ammonium saltssuch as ammonium sulfate, ammonium chloride or ammonium phosphate;organic nitrogen such as soybean hydrolysate; ammonia gas; or aqueousammonia.

It is desirable to allow required substances such as vitamin B1,L-threonine and L-tyrosine or yeast extract to be contained inappropriate amounts as organic trace nutrients. Other than the above,potassium phosphate, magnesium sulfate, iron ion, manganese ion and thelike are added in small amounts, if necessary.

Cultivation is preferably carried out under an aerobic condition for16-120 hours. The cultivation temperature is preferably controlled at25° C. to 45° C., and pH is preferably controlled at 5-8 duringcultivation. Inorganic or organic, acidic or alkaline substances as wellas ammonia gas or the like can be used for pH adjustment.

Any special techniques are not required for collecting L-methionine fromthe medium after the cultivation in the present invention. That is, thepresent invention can be practiced by a combination of well-knowntechniques such as techniques utilizing ion exchange resin andprecipitation.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will further be explained more specifically withreference to the following examples.

Example 1 Acquisition of L-Threonine Auxotrophic Strain and metJDeficient Strain from Escherichia coli W3110 Strain

<1> Preparation of Plasmid for Recombination Containing thrBC StructuralGene Having Deletion

Chromosomal DNA was prepared from W3110 strain, which was a derivativeof the wild-type K-12 strain of E. coli, by using a genomic DNApurification kit (Advanced Genetic Technology) according to theinstruction of the kit. Oligonucleotides having the nucleotide sequencesof SEQ ID NO: 1 and SEQ ID NO: 2 in Sequence Listing were synthesized.PCR was performed according to the method of Erlich et al. (PCRTechnology-Principles and Applications for DNA Amplification, ed.Erlich, H. A., Stockton Press) by using the above oligonucleotides asprimers and the aforementioned chromosomal DNA as the template toamplify a fragment of about 1 kb containing the upstream region of thethrB gene. This amplification fragment was introduced with recognitionsequences for EcoRI and SalI, which were derived from the primers. Theobtained amplified fragment was digested with restriction enzymescorresponding to the introduced recognition sites.

Similarly, PCR was performed by using oligonucleotides having thenucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 as primers toamplify a fragment of about 1 kb containing the downstream region of thethrC gene. This amplification fragment was introduced with recognitionsequences for SalI and HindIII, which were derived from the primers. Theobtained amplified fragment was digested with restriction enzymescorresponding to the introduced recognition sites. The aforementionedtwo amplified fragments, and pHSG398 (TAKARA SHUZO) digested with EcoRIand HindIII were ligated by using a ligation kit (TAKARA SHUZO), and E.coli JM109 competent cells (TAKARA SHUZO) were transformed with theligation product. Plasmids were prepared from the transformants based onthe alkaline method (Boirnboim, H. C. et al., Nucleic Acids Res., 7,1513-1523 (1979)) by using a Plasmid Extractor PI-50 (Kurabo Industries,Ltd.). From the obtained plasmids, a plasmid in which two fragments wereinserted in the EcoRI and HindIII recognition sites through SalIrecognition sites was selected based on the lengths of insertedfragments. This plasmid contained the upstream and the downstreamregions of the structural gene of thrBC, and contained a gene fragmentin which substantially full length of the structural gene of thrBC wasdeleted.

<2> Production of thrBC Structural Gene Deletion Strain by GeneticRecombination

The aforementioned plasmid and the plasmid pMAN997 having a temperaturesensitive replication origin, which was disclosed in Japanese PatentApplication No. 9-194603, were digested with EcoRI and HindIII, andligated each other. The E. coli JM109 strain was transformed with theobtained recombinant plasmid. Plasmids were extracted from thetransformants, and one having a structure where a thrBC-deleted genefragment was inserted in pMAN997 was selected, and designated aspMANΔBC. The W3110 strain was transformed with this plasmid to performgenetic recombination in a conventional manner. That is, selection ofrecombinant strains was carried out based on the L-threonine auxotrophyin M9 medium (Sambrook, J. et al., “Molecular Cloning: A LaboratoryManual/Second Edition”, Cold Spring Harbor Laboratory Press, A.3(1989)), and the obtained L-threonine auxotrophic strain was designatedas WΔBC strain.

<3> Production of metJ Deficient Strains from W3110 Strain and WΔBCStrain

Then, PCR was performed by using the W3110 strain chromosomal DNA as thetemplate and oligonucleotides having the nucleotide sequences of SEQ IDNO: 5 and SEQ ID NO: 6 as primers to amplify a fragment of about 1 kbcontaining the metB gene. This amplification fragment was introducedwith recognition sequences for EcoRI and SphI. The obtained amplifiedfragment was digested with restriction enzymes corresponding to theintroduced recognition sites.

Similarly, PCR was performed by using oligonucleotides having thenucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8 as primers toamplify a fragment of about 1 kb containing the downstream region of themetJ gene. This amplification fragment was introduced with recognitionsequences for SalI and HindIII. The obtained amplified fragment wasdigested with restriction enzymes corresponding to the introducedrecognition sites.

Then, a sequence represented as SEQ ID NO: 9, which contained SphI andHindIII recognition sites at the both ends and the promoter sequence ofthe threonine operon, and its complementary strand represented as SEQ IDNO: 10 were synthesized, annealed, and digested with restriction enzymesSphI and HindIII. The threonine promoter fragment obtained as describedabove, pHSG298 (TAKARA SHUZO) digested with EcoRI, and theaforementioned two PCR amplification fragments were mixed, and ligated.The JM109 strain was transformed with this ligation solution, andplasmids were extracted from the transformants. A plasmid comprisingligated four of the components was selected from the obtained plasmids.This plasmid had a structure where the metJ structural gene was deleted,and the promoter of metBL operon was replaced with the threoninepromoter.

The plasmid obtained above and the plasmid pMAN997 having a temperaturesensitive replication origin, which is disclosed in Japanese PatentApplication No. 9-194603, were digested with EcoRI, and ligated eachother. A plasmid having a structure where a metJ-deleted fragment wasinserted into pMAN997 was selected, and designated as pMANΔJ. The W3110strain and the WΔBC strain were transformed with this plasmid to performgenetic recombination in a conventional manner. Selection of recombinantstrains was performed based on the lengths of amplified products fromPCR utilizing DNA prepared from the cells as the template, and theoligonucleotides represented in SEQ ID NO: 6 and SEQ ID NO: 8 asprimers. The metJ-deleted strains obtained from the W3110 strain and theWΔBC strain were designated as WΔJ strain and WΔBCΔJ strain,respectively.

In order to confirm the effect of the metJ deletion by therecombination, a crude enzyme extract was prepared from the cells, andthe activities of HTS and cystathionine synthase were measured. TheW3110 strain and the WΔJ strain were each inoculated to 2 ml of LBmedium, and cultured at 37° C. overnight. 1 ml of the medium wascentrifuged at 5,000 rpm for 10 minutes, and the cells were washed twicewith 0.9% saline. The obtained cell were suspended in 1 ml of 0.9%saline, 0.5 ml of which was inoculated to 50 ml of Davis-Mingioliminimal medium (Davis B. D., and Mingioli, E. S., J. Bacteriol., 60,17-28 (1950)) containing 5 mM L-methionine. The cells were cultured at37° C. for 24 hours, then the medium was centrifuged at 8,000 rpm for 10minutes, and the cells were washed twice with 0.9% saline. The cellswere suspended in 3 ml of 50 mM potassium phosphate buffer (pH 7.5)containing 1 mM dithiothreitol. This suspension was subjected to a celldisruption treatment at 4° C. with a power of 150 W for 5 minutes byusing an ultrasonicator (Kubota Co.). The sonicated suspension wascentrifuged at 15,000 rpm for 30 minutes, and the supernatant wasdesalted in a Sephadex G-50 column (Pharmacia) to obtain a crude enzymeextract. The HTS activity and the cystathionine synthase activity in thecrude enzyme extract were measured.

As for the HTS activity, 5 μl of the crude enzyme extract was added to areaction mixture comprising 0.1 M potassium phosphate (pH 7.5), 1 mMsuccinyl-coenzyme A (Sigma), 0.2 nM DL-[¹⁴C]homoserine (MuromachiChemical Industry), and 0.2 mM L-homoserine to obtain a volume of 50 μl,and allowed to react at 30° C. for 10 minutes. 1 μl of the reactionmixture was spotted on a cellulose plate (Merck), and developed with amixed solvent containing acetone, butanol, water, and diethylamine at aratio of 10:10:5:2. After the plate was air-dried, autoradiography wasperformed by using an image analyzer (Fuji Photo Film).

The cystathionine synthase has been known to produce α-ketobutyric acid,ammonia, and succinic acid from O-succinylhomoserine in the absence ofL-cysteine, and this can be utilized for simple detection thereof(Holbrook, E. L. et al., Biochemistry, 29, 435-442 (1990)). 100 μl ofthe crude enzyme extract was added to a reaction mixture comprising 0.2M Tris-HCl (pH 8), mM O-succinylhomoserine (Sigma), and 0.25 mMpyridoxal phosphate (Sigma) to obtain a volume of 1 ml, allowed to reactat 37° C. for 20 minutes, and cooled with ice. The O-succinylhomoserinein this reaction mixture was quantitated by reverse phase HPLC (GLSciences), and the reduced amount of O-succinylhomoserines wascalculated by using a reaction mixture not added the crude enzymeextract. The reaction was also performed with no addition of pyridoxalphosphate, and pyridoxal phosphate-dependent reduction ofO-succinylhomoserine was defined to be the cystathionine synthaseactivity.

The specific activities for the HTS activity and the cystathioninesynthase activity measured as described above were shown in Table 1.While the HTS activity was hardly detected in the W3110 strain due tothe effect of L-methionine addition, marked activity was observed in theWΔJ strain. As also for the cystathionine synthase activity, remarkableincrease was observed in the WΔJ strain compared with the W3110 strain.From these results, the effects of the metJ deletion and the promotersubstitution in the metBL operon by recombination were confirmed.

TABLE 1 HTS activity and cystathionine synthase activity in metJdeficient strain Cystathionine synthase HTS activity activity Strain(mmol/min/mg protein) (mmol/min/mg protein) W3110 0.3 140 WΔJ 126 1300

Example 2 Acquisition of metK Mutant from W3110 Strain

The W3110 strain was cultured in LB medium (Sambrook, J. et al.,“Molecular Cloning: A Laboratory Manual/Second Edition”, Cold SpringHarbor Laboratory Press, A.1 (1989)) at 37° C. overnight. 1 ml of thecultured medium was centrifuged at 5,000 rpm for 10 minutes, and thecells were washed twice with 0.9% saline. The obtained cells weresuspended in 100 μl of 0.9% saline, 10 μl of which was inoculated to 5ml of Davis-Mingioli minimal medium containing 0.1 g/l of DL-norleucine(NL), and cultured at 37° C. for 5 days.

Some of the grown colonies were subjected to colony separation on LBagar medium, and their growth was confirmed again in Davis-Mingioliminimal medium containing 0.1 g/l of NL to select 12 NL-resistantstrains. Chromosomal DNA was prepared from these resistant strains. PCRwas performed by using this chromosomal DNA as a template and two sortsof primers having the sequences of SEQ ID NOS: 11 and 12 to amplify themetK gene. The nucleotide sequence of this amplification fragment wasdetermined by using amplification primers of which sequences are shownas SEQ ID NO: 11 and 12, and primers of which sequences are shown as SEQID NOS: 13, 14, 15, and 16. The nucleotide sequence determination wasperformed by using a Dye Terminator Cycle Sequencing Kit (Perkin-Elmer)on a DNA sequencer Model 373S (Perkin-Elmer) in accordance with theinstructions attached to them. The nucleotide sequence of the wildstrain W3110 determined as a control completely coincided with thesequence of metK reported by Blattner et al. (Blattner, F. R. et al.,Science, 277, 1453-1462 (1997)). This sequence is represented as SEQ IDNO: 17. Further, the amino acid sequence of SAM synthetase which may beencoded by the sequence of SEQ ID NO: 17 is shown in SEQ ID NO: 18.

Among the NL resistant strains, a mutation in the structural gene ofmetK was found in 3 strains out of the 12 strains, which were designatedas WNL2, WNL24, and WNL32. As for the metK nucleotide sequences of thesemutant strains, in the wild-type nucleotide sequence shown as SEQ ID NO:17, the WNL2 strain had replacement of adenine by cytosine at the 907thposition, the WNL24 strain had replacement of thymine by adenine at the554th position, and the WNL32 strain had deletion of cytosine at the1132nd position. As a result, it was found that, in the amino acidsequence of SAM synthetase represented as SEQ ID NO: 18, the SAMsynthetase of the WNL2 strain had replacement of isoleucine by leucineat the 303rd position, that of the WNL24 strain had replacement ofvaline by glutamic acid at the 185th position, and that of the WNL32strain had replacement of arginine at the 378th position and subsequentamino acid residues by alanine-methionine-leucine-proline-valine due todeletion of one nucleotide. It was estimated that the SAM synthetaseactivity was reduced in these strains.

Example 3 Production of L-Methionine by Introduction of metK Mutationand Amplification of Wild-Type metA Gene

(1) Introduction of metK Mutation into WΔBCΔJ Strain

PCR was performed by using each chromosomal DNA of the metK gene mutantstrains, WNL2 strain, WNL24 strain, and WNL32 strain, as a template, andoligonucleotides of SEQ ID NO: 19 and SEQ ID NO: 20 as primers toamplify a fragment of about 2.5 kb containing the metK gene. Thisamplification fragment was introduced with recognition sequences forHindIII at the both ends. The obtained amplified fragment was digestedwith HindIII. pSTV28 (TAKARA SHUZO) digested with HindIII and the PCRamplification fragment were mixed and ligated, and the JM109 strain wastransformed with the ligation product. Plasmids were extracted from thetransformants. From the obtained plasmids, plasmids inserted with thePCR amplification fragment were selected. As for these plasmids,mutations in the metK structural gene were confirmed by determiningtheir nucleotide sequences.

HindIII digestion fragments of these plasmids were each cloned intopMAN997 digested with HindIII, and the resulting plasmids weredesignated as pMANK-2, pMANK-24, and pMANK-32, respectively. The WΔBCΔJstrain was transformed with these plasmids to obtain geneticrecombination in a conventional manner. Chromosomal DNA was extractedfrom the recombinant strains, and used as a template together witholigonucleotides having the nucleotide sequences of SEQ ID NO: 11 andSEQ ID NO: 12 as primers to perform PCR, and the amplification productswere examined for nucleotide sequence to select those having mutations.The metK mutant strains obtained from the WΔBCΔJ strain were designatedas WΔBCΔJK-2 strain, WΔBCΔJK-24 strain, and WΔBCΔJK-32 strain,respectively.

(2) Amplification of metA Gene

PCR was performed by using W3110 strain chromosomal DNA as a template,and oligonucleotides having the nucleotide sequences of SEQ ID NO: 21and SEQ ID NO: 22 as primers to amplify a fragment of about 1 kbcontaining the metA gene. This amplification fragment was introducedwith recognition sequences for SphI and SalI at the both ends. Theobtained amplified fragment was digested with restriction enzymescorresponding to the introduced recognition sites. The digested productwas cloned into pHSG398 digested with SphI and SalI. The nucleotidesequence of the inserted fragment was determined by using amplificationprimers represented as SEQ ID NOS: 21 and 22, and primers having thesequences of SEQ ID NOS: 23 and 24. The determined nucleotide sequenceof metA of the wild strain W3110 completely coincided with the sequenceof metA reported by Blattner et al. (Blattner, F. R. et al., Science,277, 1453-1462 (1997)). This sequence is represented as SEQ ID NO: 25.Further, the amino acid sequence of HTS which may be encoded by thesequence of SEQ ID NO: 25 is shown as SEQ ID NO: 26.

A SphI and SalI digestion product of this plasmid, HindIII and SphIdigestion product of the threonine promoter of Example 1, and pMW118(Nippon Gene) digested with HindIII and SalI were mixed and ligated. TheJM109 strain was transformed with this reaction mixture, and plasmidswere extracted from the transformants. From the obtained plasmids, aplasmid in which the three components were ligated was selected. Thisplasmid had a structure where the metA gene was positioned at thedownstream from the threonine promoter, by which the metA was expressed.This plasmid was designated as pMWPthmetA-W. The W3110 strain, WΔBCstrain, WΔBCΔJ strain, WΔBCΔJK-2 strain, WΔBCΔJK-24 strain, andWΔBCΔJK-32 strain were transformed with this plasmid to obtaintransformants.

Each transformant was cultured at 37° C. overnight on an LB platecontaining 50 mg/l of ampicillin. The cells were inoculated to 20 ml ofmedium at pH 7 containing 40 g/l of glucose, 1 g/l of magnesium sulfate,16 g/l of ammonium sulfate, 1 g/l of potassium dihydrogenphosphate, 2g/l of yeast extract (Bacto Yeast-Extract, Difco), 0.01 g/l of manganesesulfate, 0.01 g/l of iron sulfate, 30 g/l of calcium carbonate, 50 mg/lof ampicillin, and 0.5 g/l of L-threonine, and cultured at 37° C. for 48hours.

The cells were separated from the culture, and the amount ofL-methionine was measured by an amino acid analyzer (Hitachi). Theresults are shown in Table 2. L-Methionine, which was not detected forthe W3110 strain, was increased in the WΔBC strain and the WΔBCΔJstrain. Concerning the metK mutant strains, while the amount ofL-methionine decreased in the WΔBCΔJK-2 strain, a comparable amount wasobserved in the WΔBCΔJK-32 strain, and the amount was increased in theWΔBCΔJK-24 strain. Thus, the effect on the L-methionine production wasobserved. The WΔBCΔJK-24 strain harboring the plasmid pMWPthrmetA-W wasgiven a private number AJ13425, and it was deposited at the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, Ministry of International Trade and Industry(postal code 305-8566, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken,Japan) on May 14, 1998 as an accession number of FERM P-16808, andtransferred from the original deposit to international deposit based onBudapest Treaty on Sep. 27, 1999, and has been deposited as depositionnumber of FERM BP-6895.

TABLE 2 L-methionine production amount of wild-type metA introducedstrains Production amount Strain (g/l) W3110/pMWPthrmetA-W (metA^(e))0.000 WΔBC/pMWPthrmetA-W (thrBC⁻, metA^(e)) 0.008 WΔBCΔJ/pMWPthrmetA-W0.022 (metBL^(e), thrBC⁻, metA^(e)) WΔBCΔJK-2/pMWPthrmetA-W 0.014(thrBC⁻, metJ⁻, metBL^(e), metK¹, metA^(e)) WΔBCΔJK-24/pMWPthrmetA-W0.141 (thrBC⁻, metJ⁻, metBL^(e), metK¹, metA^(e))WΔBCΔJK-32/pMWPthrmetA-W 0.023 (thrBC⁻, metJ⁻, metBL^(e), metK¹,metA^(e)) metK¹: reduced metK, metA^(e): enhanced metA, metBL^(e):enhanced metBL

Example 4 Acquisition of metA Mutant Strain and Inhibition-DesensitizedType metA Gene

The W3110 strain was inoculated to 2 ml of LB medium, and cultured at37° C. for 8 hours. 1 ml of the medium was centrifuged at 5,000 rpm for10 minutes, and the cells were washed twice with 0.9% saline. Theobtained cells were suspended in 100 μl of 0.9% saline, 5 μl of whichwas inoculated to 5 ml of Davis-Mingioli minimal medium containing 1 g/lof α-methyl-DL-methionine (MM), and cultured at 37° C. for 3 days. Themedium was properly diluted, plated on Davis-Mingioli minimal mediumcontaining 1 g/l of MM, and cultured at 37° C. overnight. Some of growncolonies were subjected to colony separation on LB agar medium, andtheir growth was confirmed again on Davis-Mingioli minimal mediumcontaining 1 g/l of MM. This procedure was independently performed 9times, and six independent resistant strains, each designated as WMM4,WMM5, WMM6, WMM7, WMM8, and WMM9, were obtained.

Chromosomal DNA was prepared from these resistant strains. PCR wasperformed by using each chromosomal DNA as a template and primers havingthe sequences represented as SEQ ID NOS: 21 and 22 to amplify the metAgene. The nucleotide sequence of each amplification fragment wasdetermined by using primers for amplification of which nucleotidesequences are represented as SEQ ID NOS: 21 and 22, and primers havingthe sequences represented as SEQ ID NOS: 23 and 24. As for the metAnucleotide sequence of the resistance strains, in the nucleotidesequence of wild-type metA represented as SEQ ID NO: 25, thymine at theposition of 887 was changed to guanine in the WMM4 strain, cytocine atthe position of 893 was changed to thymine in the WMM5 strain, was thewild-type sequence was found in the WMM6 strain, a sequence of ATCTCcorresponding the 886th to the 890th nucleotides iterated twice, and aninsertion sequence consisting of about 1300 nucleotides, called IS2(Ghosal, D. et al., Nucleic Acids Res., 6, 1111-1122 (1979)), waspresent between the repeated sequences in the WMM7 and WMM8 strains, andcytosine at the position of 79 was changed to thymine in the WMM9strain. As a result, it was found that, in the amino acid sequence ofHTS represented as SEQ ID NO: 26, the 296th isoleucine was changed toserine in the WMM4 strain, proline at the position of 298 was changed toleucine in the WMM5 strain, proline at the position of 298 andsubsequent amino acid residues were changed to a sequence ofarginine-leucine-alanine-proline due to an insertion sequence in theWMM7 and WMM8 strains, and arginine at the position of 27 was changed tocysteine in the WMM9 strain.

The strains WMM4, WMM5, WMM9 and WMM7, in which a mutation was observedin the metA structural gene, were each cultured in LB medium containedin a test tube at 37° overnight, and 1 ml of the medium was centrifugedat 5,000 rpm for 10 minutes. The cells were washed twice with 1 ml of0.9% saline, and suspended in 1 ml of 0.9% saline, 0.5 ml of which wasinoculated to 50 ml of minimal medium and cultured at 37° C. for oneday. The medium was centrifuged at 8,000 rpm for 10 minutes, and thecells were washed twice with 1 ml of 0.9% saline. The obtained cellswere suspended in 3 ml of 50 mM potassium phosphate buffer (pH 7.5)containing 1 mM dithiothreitol, and a crude enzyme extract was obtainedin the same manner as in Example 1. The HTS activity in the crude enzymeextract was measured with the reaction composition mentioned in Example1 in the presence of an inhibitor. The results are shown in Table 3. Theactivity was undetectable for the WMM7 strain, and this was consideredto reflect the marked decrease of the specific activity due to the aminoacid sequence change caused by the insertion sequence. The specificactivities of the other strains were about ¼ of the wild strain. Theinhibition by MM was canceled in all of the WMM4, WMM5, and WMM9strains, and the inhibition by L-methionine was also reducedconsiderably. While the inhibition by SAM was hardly canceled in theWMM9 strain, tendency of cancellation was observed in the WMM4 and WMM5strains. Inhibition by the combination of L-methionine and SAM, whichexhibited the strongest inhibition for the wild-type HTS activity, wasalso markedly reduced in the WMM4 and WMM5 strains

TABLE 3 Activity of HTS derived from MM resistant strains in thepresence of various inhibitors HTS activity (mmol/min/mg protein)Inhibitor W3110 WMM9 WMM4 WMM5 WMM7 No addition 22.3 5.0 4.5 4.5 0.0 0.1mM MM 18.6 4.9 4.1 4.6 0.0 1 mM MM 7.0 2.7 4.6 4.8 0.0 0.1 mM Met 14.32.5 4.5 4.2 0.0 1 mM Met 0.8 2.2 4.0 4.0 0.0 0.1 mM SAM 17.0 1.1 4.6 3.60.0 1 mM SAM 3.0 0.5 2.6 3.3 0.0 0.1 mM SAM + 0.0 0.9 5.6 2.8 0.0 0.1 mMMet

Example 5 L-Methionine Production by Introduction of Mutant Meta

PCR was performed by using chromosomal DNA from each of the WMM9, WMM4and WMM5 strains among the metA mutants obtained in Example 4 as atemplate, and oligonucleotides having the sequences of SEQ ID NO: 21 andSEQ ID NO: 22 as primers to amplify a fragment containing the metA gene.This amplification fragment had recognition sequences for SphI and SalIat the both ends. The both ends of this amplified fragment were digestedwith SphI and SalI, and cloned into pHSG398 digested with SphI and SalI.The nucleotide sequence of each insert fragment was determined toconfirm the mutation point. A SphI and SalI digestion product of thisplasmid, HindIII and SphI digestion product of the threonine promotermentioned in Example 1, and pMW118 (Nippon Gene) digested with HindIIIand SalI were mixed and ligated. The JM109 strain was transformed withthis ligation solution, and plasmids were extracted from thetransformants. From the obtained plasmids, those comprising ligatedthree components were selected, and designated as pMWPthrmetA-9,pMWPthrmetA-4, and pMWPthrmetA-5, respectively.

Further, in order to obtain combination of the mutation points of eachmutant metA gene, site-specific mutagenesis was performed by usingMutan-Super Express Km (TAKARA SHUZO) according to the instruction ofthe manufacturer. pMwPthrmetA-9+4 were produced by combining the metA-4mutation with the metA-9 mutation using an oligonucleotide having thesequence of SEQ ID NO: 27. pMWPthrmetA-9+5 was similarly produced bycombining the metA-5 mutation with the metA-9 mutation. Furthermore,pMWPthrmetA-9+4+5 was produced by combining the metA-9 mutation with themetA-4 and metA-5 mutations using an oligonucleotide having the sequenceof SEQ ID NO: 28.

The WΔBCΔJK-32 strain was transformed with these plasmids to obtaintransformants. Each of the transformants was cultured at 37° C.overnight on an LB plate containing 50 mg/l of ampicillin. The cellswere inoculated to 20 ml of medium at pH 7 containing 40 g/l of glucose,1 g/l of magnesium sulfate, 16 g/l of ammonium sulfate, 1 g/l ofpotassium dihydrogenphosphate, 2 g/l of yeast extract (BactoYeast-Extract, Difco), 0.01 g/l of manganese sulfate, 0.01 g/l of ironsulfate, 30 g/l of calcium carbonate, 50 mg/l of ampicillin, and 0.5 g/lof L-threonine, and cultured at 37° C. for 48 hours. The cells wereseparated from the culture, and the amount of L-methionine was measuredby an amino acid analyzer (Hitachi). The results are shown in Table 4.The amount of L-methionine accumulation increased several times in thestrains introduced with a mutant metA, compared with the strainintroduced with a wild-type metA. Furthermore, by combining mutations,further increase of L-methionine production amount was obtained.

TABLE 4 L-methionine production amount of mutant metA-introduced strainsAmount of L-methionine Strain production (g/l) WΔBCΔΔJK-32/pMWPthrmetA-W0.023 WΔBCΔJK-32/pMWPthrmetA-9 0.158 WΔBCΔJK-32/pMWPthrmetA-4 0.108WΔBCΔJK-32/pMWPthrmetA-5 0.131 WΔBCΔJK-32/pMWPthrmetA-9 + 4 0.206WΔBCΔJK-32/pMWPthrmetA-5 + 9 0.207 WΔBCΔJK-32/pMWPthrmetA-9 + 4 + 50.236

1. A method for producing L-methionine which comprises culturing arecombinant Escherichia bacterium in a medium to produce and accumulateL-methionine in the medium in an amount in excess of the correspondingunmodified Escherichia bacterium, and collecting the L-methionine fromthe medium, wherein the bacterium is deficient in repressor ofL-methionine biosynthesis system encoded by the endogenous metJ gene andhas L-methionine productivity, activity of intracellular homoserinetranssuccinylase encoded by the metA gene of a Escherichia bacterium isincreased compared to an unmodified Escherichia bacterium by increasingcopy number of the metA gene including its own promoter, or replacingthe native promoter with a stronger promoter, and the bacteriumcomprises at least one characteristic selected from the group consistingof: (a) exhibits reduced activity of intracellular S-adenosylmethioninesynthetase encoded by the endogenous metK gene as compared to anunmodified Escherichia bacterium; (b) exhibits L-threonine auxotrophy;(c) exhibits enhanced activity of intracellular cystathionine γ-synthaseencoded by the metB gene of a Escherichia bacterium and enhancedactivity of intracellular aspartokinase-homoserine dehydrogenase IIencoded by the metL gene of a Escherichia bacterium as compared to anunmodified Escherichia bacterium by increasing copy number of each ofthe genes including their own promoters, or replacing the nativepromoter with a stronger promoter; and (d) has a homoserinetranssucinylase for which concerted inhibition by L-methionine andS-adenosylmethionine is desensitized, wherein the homoserinetranssuccinylase comprising the amino acid sequence of SEQ ID NO: 26contains at least one amino acid replacement wherein said at least oneamino acid replacement is independently selected from the groupconsisting of replacement of the amino acid residue Arg-27 withcysteine, replacement of the amino acid residue Ile-296 with serine, andreplacement of the amino acid residue Pro-298 with leucine.
 2. Themethod according to claim 1, wherein the bacterium is Escherichia coli.3. The method according to claim 1, wherein the bacterium comprises atleast the characteristic (a).
 4. The method according to claim 1,wherein the bacterium comprises at least the characteristic (b).
 5. Themethod according to claim 1, wherein the bacterium comprises at leastthe characteristic (c).
 6. The method according to claim 1, wherein thebacterium comprises at least the characteristic (d).
 7. The methodaccording to claim 1, wherein the bacterium comprises thecharacteristics (a) and (b).
 8. The method according to claim 1, whereinthe bacterium comprises the characteristics (a) and (c).
 9. The methodaccording to claim 1, wherein the bacterium comprises thecharacteristics (a) and (d).
 10. The method according to claim 1,wherein the bacterium comprises the characteristics (b) and (c).
 11. Themethod according to claim 1, wherein the bacterium comprises thecharacteristics (b) and (d).
 12. The method according to claim 1,wherein the bacterium comprises the characteristics (c) and (d).
 13. Themethod according to claim 1, wherein the bacterium comprises thecharacteristics (a), (b), and (c).
 14. The method according to claim 1,wherein the bacterium comprises the characteristics (a), (b), and (d).15. The method according to claim 1, wherein the bacterium comprises thecharacteristics (a), (c), and (d).
 16. The method according to claim 1,wherein the bacterium comprises the characteristics (b), (c), and (d).17. The method according to claim 1, wherein the bacterium comprises thecharacteristic (a), (b), (c), and (d).
 18. The method according to claim1, wherein the activity of intracellular S-adenosylmethionine synthetaseis reduced due to that the bacterium has S-adenosylmethionine synthetasewhich contains amino acid substitution which is selected from the groupconsisting of replacement of the amino acid residue Ile-303 withleucine, replacement of the amino acid residue Val-185 with glutamicacid, and replacement of amino acid residues 378-384 with the amino acidsequence of SEQ ID NO: 29, respectively in the amino acid sequence ofSEQ ID NO:
 18. 19. The method according to claim 3, wherein the activityof intracellular S-adenosylmethionine synthetase is reduced due to thatthe bacterium has S-adenosylmethionine synthetase which contains aminoacid substitution which is selected from the group consisting ofreplacement of the amino acid residue Ile-303 with leucine, replacementof the amino acid residue Val-185 with glutamic acid, and replacement ofamino acid residues 378-384 with the amino acid sequence of SEQ ID NO:29, respectively in the amino acid sequence of SEQ ID NO:
 18. 20. Themethod according to claim 1, wherein the L-threonine auxotrophy is dueto deletion of the thrBC genes.
 21. The method according to claim 4,wherein the L-threonine auxotrophy is due to deletion of the thrBCgenes.